Process for the preparation of L-amino acids via overexpression of the PTSH gene

ABSTRACT

The invention relates to an isolated polynucleotide containing a polynucleotide sequence selected from the group comprising
     a) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide containing the amino acid sequence of SEQ ID no. 2,   b) polynucleotide which codes for a polypeptide containing an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID no.2,   c) polynucleotide which is complementary to the polynucleotides of a) or b), and   d) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c),
 
and a process for the fermentative production of L-amino acids with enhancement of the ptsH gene coding for component H of the phosphotransferase system, and the use of the above polynucleotides as primer or hybridisation probe.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 09/819,930,filed Mar. 29, 2001, now U.S. Pat. No. 6,818,432, which is acontinuation-in-part of U.S. patent application Ser. No. 09/755,187filed Jan. 8, 2001, now abandoned which is a continuation-in-part ofU.S. patent application Ser. No. 09/503,189, filed on Feb. 14, 2000, nowabandoned which claims priority to German Patent Appl. No. 100 01 101.2,filed Jan. 13, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides nucleotide sequences coding for ptsH andprocesses for the fermentative preparation of L-amino acids,particularly L-lysine, in which the ptsH gene is enhanced, usingcoryneform bacteria.

2. Background Information

L-amino acids, particularly L-lysine, are used in human medicine and inthe pharmaceutical industry, and particularly in animal nutrition.

It is known to prepare L-amino acids by fermentation of strains ofcoryneform bacteria, particularly Corynebacterium glutamicum. In view ofthe great importance, work is constantly being carried out to improvethe preparation processes. Process improvements may relate to measuresinvolving the fermentation technique, such as, e.g., agitation andoxygen supply, or the composition of the nutrient media such as, e.g.,the sugar concentration during fermentation, or the work up to theproduct form by, e.g., ion exchange chromatography, or the intrinsicperformance properties of the microorganism itself.

In order to improve the performance properties of said microorganisms,methods of mutagenesis, selection and mutant selection are employed.Strains thereby obtained are resistant to antimetabolites such as, e.g.,the lysine analogue S-(2-aminoethyl) cysteine, or auxotrophic formetabolites of regulatory importance and produce L-lysine.

For some years, methods of recombinant DNA technology have also beenused to improve strains of coryneform bacteria producing L-amino acidsby amplifying individual biosynthesis genes for L-amino acids andexamining the effect on L-amino acid production. Review articles on thissubject may be found inter alia in Kinoshita (“Glutamic Acid Bacteria”,in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.),Benjamin Cummings, London, UK, 1985, 115–142), Hilliger (BioTec 2, 40–44(1991)), Eggeling (Amino Acids 6:261–272 (1994)), Jetten and Sinskey(Critical Reviews in Biotechnology 15, 73–103 (1995)) and Sahm et al.(Annuals of the New York Academy of Science 782, 25–39 (1996)).

SUMMARY OF THE INVENTION Object of the Invention

The inventors set themselves the task of providing new measures for theimproved fermentative preparation of L-amino acids, particularlyL-lysine.

Description of the Invention

L-amino acids, particularly L-lysine, are used in human medicine, in thepharmaceutical industry and particularly in animal nutrition. It is ofgeneral interest, therefore, to provide new improved processes for thepreparation of L-amino acids, particularly L-lysine.

Where the terms L-lysine or lysine are mentioned below, they refer notonly to the base but also to the salts such as, e.g., lysinemonohydrochloride or lysine sulfate.

The invention provides an isolated polynucleotide from coryneformbacteria containing a polynucleotide sequence selected from the groupcomprising

-   a) polynucleotide which is at least 70% identical to a    polynucleotide coding for a polypeptide which contains the amino    acid sequence of SEQ ID no. 2,-   b) polynucleotide which codes for a polypeptide containing an amino    acid sequence which is at least 70% identical to the amino acid    sequence of SEQ ID no.2,-   c) polynucleotide which is complementary to the polynucleotides    of a) or b), and-   d) polynucleotide containing at least 15 successive nucleotides of    the polynucleotide sequence of a), b) or c).

The invention also provides a polynucleotide which is a DNA, preferablyrecombinant, which can be replicated in coryneform bacteria.

The invention also provides a polynucleotide which is an RNA.

The invention also provides a polynucleotide which is preferably areplicable DNA containing:

-   (i) the nucleotide sequence shown in SEQ ID no.1, or-   (ii) at least one sequence which corresponds to the sequence (i)    within the degeneracy region of the genetic code, or-   (iii) at least one sequence which hybridises with the sequence    complementary to sequence (i) or (ii), and optionally-   (iv) functionally neutral sense mutations in (i).

The invention also provides

a vector containing one of the polynucleotides mentioned,and coryneformbacteria acting as host cell which contain the vector.

The invention also provides polynucleotides comprising substantially apolynucleotide sequence which may be obtained by screening byhybridising an appropriate gene bank containing the complete gene withthe polynucleotide sequence corresponding to SEQ ID no. 1, with a probewhich contains the sequence of the above-mentioned polynucleotideaccording to SEQ ID no. 1 or a fragment thereof, and isolating the DNAsequence mentioned.

Polynucleotide sequences according to the invention are suitable ashybridisation probes for RNA, cDNA and DNA, for isolating full-lengthcDNA which code for component H of the phosphotransferase system (ptsH)and for isolating those cDNA or genes which have great similarity ofsequence with that of the gene for component H of the phosphotransferasesystem.

Polynucleotide sequences according to the invention are also suitable asprimers for the preparation of DNA of genes which code for component Hof the phosphotransferase system by the polymerase chain reaction (PCR).

The oligonucleotides acting as probes or primers contain at least 30,preferably at least 20, more particularly preferably at least 15successive nucleotides. oligonucleotides with a length of at least 40 or50 nucleotides are also suitable.

“Isolated” means separated from its natural surroundings.

“Polynucleotide” refers generally to polyribonucleotides andpolydeoxyribonucleotides, which may be unmodified RNA or DNA or modifiedRNA or DNA.

The term “polypeptides” means peptides or proteins which contain two ormore amino acids bound by way of peptide bonds.

The polypeptides according to the invention include a polypeptideaccording to SEQ ID no. 2, and also those with the biological activityof component H of the phosphotransferase system and also those which areat least 70% identical to the polypeptide according to SEQ ID no. 2,preferably at least 80% and in particular those which are 90% to 95%identical to the polypeptide according to SEQ ID no. 2 and have theactivity mentioned.

The invention also relates to a process for the fermentative preparationof L-amino acids, particularly L-lysine, using coryneform bacteria whichin particular already produce an L-amino acid and in which thenucleotide sequences coding for the ptsH gene are enhanced, particularlyoverexpressed.

The term “enhancement” describes in this context the increase inintracellular activity of one or more enzymes in a microorganism whichare coded for by the corresponding DNA, by, for example, increasing thecopy number of the gene or genes or alleles, using a strong promotor orusing a gene or allele which codes for a corresponding enzyme with ahigh activity and optionally combining said measures.

The microorganisms which are the subject of the present invention mayproduce L-amino acids, particularly L-lysine from glucose, sucrose,lactose, fructose, maltose, molasses, starch, cellulose or from glyceroland ethanol. They may be representatives of coryneform bacteria,particularly of the Corynebacterium genus. A particular example of theCorynebacterium genus is the Corynebacterium glutamicum type which isknown by experts to have the ability to produce L-amino acids.

Examples of suitable strains of the Corynebacterium genus,particularlyof the Corynebacterium glutamicum type include the well known wild-typestrains

-   -   Corynebacterium glutamicum ATCC13032    -   Corynebacterium acetoglutamicum ATCC15806    -   Corynebacterium acetoacidophilum ATCC13870    -   Corynebacterium thermoaminogenes FERM BP-1539    -   Corynebacterium melassecola ATCC17965    -   Brevibacterium flavum ATCC14067    -   Brevibacterium lactofermentum ATCC13869 and    -   Brevibacterium divaricatum ATCC14020

and L-lysine-producing mutants and strains prepared therefrom, such as,for example

-   -   Corynebacterium glutamicum FERM-P 1709    -   Brevibacterium flavum FERM-P 1708    -   Brevibacterium lactofermentum FERM-P 1712    -   Corynebacterium glutamicum FERM-P 6463    -   Corynebacterium glutamicum FERM-P 6464 and    -   Corynebacterium glutamicum DSM5715.

The inventors succeeded in isolating from C.glutamicum the new ptsH genecoding for component H of the phosphotransferase system.

In order to isolate the ptsH gene or other genes from C. glutamicum, agene bank of this microorganism is first prepared in E. coli. Thepreparation of gene banks is documented in generally known textbooks andmanuals. Examples include the textbook by Winnacker: Gene und Klone,Eine Einführung in die Gentechnologie (Verlag Chemie, Weinheim, Germany,1990) or the manual by Sambrook et al.: Molecular Cloning, A LaboratoryManual (Cold Spring Harbor Laboratory Press, 1989). A very well knowngene bank is that of the E. coli K-12 strain W3110, which was preparedby von Kohara et al. (Cell 50, 495–508 (1987)) in λ-vectors. Bathe etal. (Molecular and General Genetics, 252:255–265, 1996) describe a genebank of C. glutamicum ATCC13032 which was prepared using the cosmidvector. SuperCos I (Wahl et al., 1987, Proceedings of the NationalAcademy of Sciences USA, 84:2160–2164) in the E. coli K-12 strain NM554(Raleigh et al., 1988, Nucleic Acids Research 16:1563–1575). Börmann etal. (Molecular Microbiology 6(3), 317–326 (1992)) in turn describe agene bank of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn andCollins, Gene 11, 291–298 (1980)). In order to prepare a gene bank of C.glutamicum in E. coli, it is also possible to use plasmids such aspBR322 (Bolivar, Life Sciences, 25, 807–818 (1979)) or pUC9 (Vieira etal., 1982, Gene, 19:259–268). Particularly suitable hosts are E. colistrains which are restriction- and recombination-defective. An exampleof these is the DH5αMCR strain which was described by Grant et al.(Proceedings of the National Academy of Sciences USA, 87 (1990)4645–4649). The long DNA fragments cloned using cosmids may then in turnbe subcloned into common vectors suitable for sequencing, and thensequenced, as described in Sanger et al. (Proceedings of the NationalAcademy of Sciences of the United States of America, 74:5463–5467,1977).

The new DNA sequence coding for ptsH was obtained in this way from C.glutamicum and, as SEQ ID no. 1, forms part of the present invention.Moreover, the amino acid sequence of the corresponding protein wasderived from the present DNA sequence with the methods described above.The resulting amino acid sequence of the ptsH gene product is shown inSEQ ID no. 2.

Coding DNA sequences resulting from SEQ ID No. 1 due to the degeneracyof the genetic code also form part of the invention. Experts are alsofamiliar with conservative amino acid exchanges such as, e.g., theexchange of glycine for alanine or of aspartic acid for glutamic acid inproteins as “sense mutations” which do not lead to a fundamental changein the activity of the protein, i.e. which are functionally neutral. Itis also known that changes at the N and/or C end of a protein do notsubstantially impair or may even stabilise its function. Experts mayfind details on this subject, inter alia, in Ben-Bassat et al. (Journalof Bacteriology 169:751–757 (1987)), in O'Regan et al. (Gene 77:237–251(1989)), in Sahin-Toth et al. (Protein Sciences 3:240–247 (1994)), inHochuli et al. (Bio/Technology 6:1321–1325 (1988)) and in well knowntextbooks of genetics and molecular biology. Amino acid sequences whichare obtained in corresponding manner from SEQ ID no. 2 and these DNAsequences encoding amino acid sequences also form part of the invention.

Similarly, DNA sequences which hybridise with SEQ ID no. 1 or parts ofSEQ ID no. 1 form part of the invention. Finally, DNA sequences whichare prepared by the polymerase chain reaction (PCR) using primersobtained from SEQ ID no. 1 form part of the invention. Sucholigonucleotides typically have a length of at least 15 nucleotides.

The expert may find instructions for the identification of DNA sequencesby hybridisation inter alia in the manual “The DIG System Users Guidefor Filter Hybridization” from Firma Boehringer Mannheim GmbH (Mannheim,Germany, 1993) and in Liebl et al. (International Journal of SystematicBacteriology (1991) 41: 255–260). The expert may find instructions forthe amplification of DNA sequences using the polymerase chain reaction(PCR) inter alia in the manual by Gait: Oligonucleotide synthesis: apractical approach (IRL Press, Oxford, UK, 1984) and in Newton andGraham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

The inventors discovered that coryneform bacteria produce L-amino acids,particularly L-lysine, in an improved manner after overexpression of theptsH gene.

In order to obtain overexpression, the copy number of the correspondinggene may be increased, or the promotor and regulatory region or theribosome binding site situated upstream of the structural gene may bemutated. Expression cassettes which are incorporated upstream of thestructural gene act in the same way. As a result of inducible promoters,it is also possible to increase expression in the course of fermentativeL-amino acid production. Expression is also improved by measures toprolong the life of the m-RNA. Moreover, by preventing the degradationof the enzyme protein, the enzyme activity is also increased. The genesor gene constructs may either be present in plasmids with a differentcopy number, or integrated in the chromosome and amplified.Alternatively, overexpression of the genes concerned may be achieved byaltering the composition of the medium and the way in which the cultureis carried out.

The expert may find instructions on this subject inter alia in Martin etal. (Bio/Technology 5, 137–146 (1987)), in Guerrero et al. (Gene 138,35–41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428–430 (1988)),in Eikmanns et al. (Gene 102, 93–98 (1991)), in the European patent EPS0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Pühler(Bio/Technology 9, 84–87 (1991), in Reinscheid et al. (Applied andEnvironmental Microbiology 60, 126–132 (1994)), in LaBarre et al.(Journal of Bacteriology 175, 1001–1007 (1993)), in the patentapplication WO 96/15246, in Malumbres et al. (Gene 134, 15–24 (1993)),in the Japanese specification JP-A-10-229891, in Jensen and Hammer(Biotechnology and Bioengineering 58, 191–195 (1998)), in Makrides(Microbiological Reviews 60:512–538 (1996)) and in well known textbooksof genetics and molecular biology.

By way of example, the ptsH gene according to the invention wasoverexpressed using plasmids.

Suitable plasmids are those which are replicated in coryneform bacteria.Numerous well known plasmid vectors such as, e.g., pZ1 (Menkel et al.,Applied and Environmental Microbiology (1989) 64: 549–554), pEKEx1(Eikmanns et al., Gene 102:93–98 (1991)) or pHS2-1 (Sonnen et al., Gene107:69–74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 orpGA1. Other plasmid vectors such as, e.g., those based on pCG4 (U.S.Pat. No. 4,489,160) or pNG2 (Serwold-Davis et al., FEMS MicrobiologyLetters 66, 119–124 (1990)) or pAG1 (U.S. Pat. No. 5,158,891) may beused in the same way.

Other suitable plasmid vectors include those by means of which theprocess of gene amplification by integration into the chromosome may beemployed, as was described, e.g., by Reinscheid et al. (Applied andEnvironmental Microbiology 60, 126–132 (1994)) for the duplication andamplification of the hom-thrB operon. In this method, the complete geneis cloned into a plasmid vector which is able to replicate in a host(typically E. coli), but not in C. glutamicum. Examples of suitablevectors include pSUP301 (Simon et al., Bio/Technology 1, 784–791(1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69–73 (1994)),pGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman(1994). Journal of Biological Chemistry 269:32678–84; U.S. Pat. No.5,487,993), pCR®Blunt (Firma Invitrogen, Groningen, Niederlande; Bernardet al., Journal of Molecular Biology, 234: 534–541 (1993)) or pEM1(Schrumpf et al, 1991, Journal of Bacteriology 173:4510–4516). Theplasmid vector which contains the gene to be amplified is thentransferred by conjugation or transformation into the desired strain ofC. glutamicum. The conjugation method is described, for example, inSchäfer et al. (Applied and Environmental Microbiology 60, 756–759(1994)). Methods of transformation are described, for example, inThierbach et al. (Applied Microbiology and Biotechnology 29, 356–362(1988)), Dunican and Shivnan (Bio/Technology 7, 1067–1070 (1989)) andTauch et al. (FEMS Microbiological Letters 123, 343–347 (1994)). Afterhomologous recombination using a “cross over” event, the resultingstrain contains at least two copies of the gene concerned.

The invention also provides, therefore, a process for the fermentativepreparation of L-amino acids, particularly L-lysine, wherein a straintransformed with a plasmid vector is used and the plasmid vector carriesthe nucleotide sequence of the gene coding for component H of thephosphotransferase system.

Moreover, it was found that by exchanging the amino acid L-alanine inposition 25 of the protein component H of the phosphotransferase system(see SEQ ID NO:2) for any other proteinogenic amino acid, particularlyL-threonine (see SEQ ID NO:4), with the exception of. L-alanine,enhancement takes place and coryneform bacteria which bear thecorresponding amino acid exchange produce L-lysine in an improvedmanner. The exchange of L-alanine for L-threonine in position 25 of theamino acid sequence may be carried out preferably by exchanging thenucleobase adenine in position 235 for guanine, as shown in thenucleotide sequence according to SEQ ID NO:3.

Conventional mutagenesis methods using mutagenic substances such as, forexample, N-methyl-N′-nitro-N-nitrosoguanidine or ultraviolet light maybe used for mutagenesis. Moreover, in vitro methods such as, forexample, a treatment with hydroxylamine or mutagenic oligonucleotides orthe polymerase chain reaction (PCR) may be used for mutagenesis.

Accordingly, the invention also provides coryneform bacteria whichcontain a protein component H of the phosphotransferase system in whichthe amino acid sequence in position 25 shown under SEQ ID NO:2 isexchanged for another amino acid except L-alanine. A further aspect ofthis invention is coryneform bacteria which contain a correspondingprotein in which the amino acid L-alanine in position 25 of the protein(see SEQ ID NO:2) is exchanged for L-threonine (see SEQ ID NO:4).

Accordingly, the invention also provides polynucleotide sequencesoriginating from coryneform bacteria which contain genes or alleleswhich encode the above-mentioned protein components H of thephosphotransferase system.

The present invention also provides coryneform bacteria which contain aDNA encoding a protein component H of the phosphotransferase systemwhich is characterised by the exchange of L-alanine for L-threonine inposition 25, which DNA contains adenine instead of the nucleobaseguanine in position 235 (see SEQ ID NO:1), as shown in SEQ ID NO:3:

In addition, it may be advantageous for the preparation of L-aminoacids, particularly L-lysine, to enhance not only the ptsH gene but alsoother genes of the biosynthesis pathway of the desired L-amino acid sothat one or more enzymes of the biosynthesis pathway in question,glycolysis, anaplerotic reactions or amino acid export, isoverexpressed.

For the preparation of L-lysine, for example, it is possible tooverexpress simultaneously one or more of the genes selected from thegroup comprising

-   the dapA gene coding for dihydrodipicolinate synthase (EP-B 0 197    335),-   the gap gene coding for glyceraldehyde-3-phosphate dehydrogenase    (Eikmanns (1992), Journal of Bacteriology 174:6076–6086),-   the tpi gene coding for triosephosphate isomerase (Eikmanns (1992),    Journal of Bacteriology 174:6076–6086),-   the pgk gene coding for 3-phosphoglycerate kinase (Eikmanns (1992),    Journal of Bacteriology 174:6076–6086),-   the ptsM gene coding for component M of the    phosphoenolpyruvate-sugar-phosphotransferase system (ptsM) (Lee et    al. (1994), FEMS Microbiology Letters 1–2, 137–145),-   the pyc gene coding for pyruvate carboxylase (DE-A-198 31 609), and-   the lysE gene coding for lysine export (DE-A-195 48 222).

Moreover, for the production of L-amino acids, particularly L-lysine, itmay be advantageous, in addition to the ptsH gene, simultaneously toattenuate

-   the pck gene coding for phosphoenolpyruvate carboxykinase (DE 199 50    409.1, DSM 13047) and/or-   the pgi gene coding for glucose-6-phosphate isomerase (U.S. Ser. No.    09/396,478, DSM 12969)-   the poxB gene coding for pyruvate oxidase (DE 19846499.1; DSM    13114).

Moreover, for the production of L-amino acids, particularly L-lysine, itmay be advantageous, in addition to the overexpression of the ptsH gene,to exclude unwanted side reactions (Nakayama: “Breeding of Amino AcidProducing Micro-organisms”, in: Overproduction of Microbial Products,Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The microorganisms produced according to the invention-may be cultivatedcontinuously or batchwise in the batch process (batch cultivation) or inthe fed-batch or repeated fed-batch process in order to produce L-aminoacids, particularly L-lysine. Summaries of well known cultivationmethods are described in the textbook by Chmiel (Bioprozesstechnik 1.Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag,Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren undperiphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must satisfy the requirements of thestrains concerned in a suitable manner. Descriptions of culture media ofvarious microorganisms are contained in the manual “Manual of Methodsfor General Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981). Suitable sources of carbon include sugarsand carbohydrates such as, e.g., glucose, sucrose, lactose, fructose,maltose, molasses, starch and cellulose, oils and fats such as, e.g.,soyabean oil, sunflower oil, groundnut oil and coconut fat, fatty acidssuch as, e.g., palmitic acid, stearic acid and linoleic acid, alcoholssuch as, e.g., glycerol and ethanol and organic acids such as, e.g.,acetic acid. Said substances may be used individually or as mixtures.Suitable sources of nitrogen include organic nitrogen-containingcompounds such as peptones, yeast extract, meat extract, malt extract,maize swelling water, soyabean flour and urea or inorganic compoundssuch as ammonium sulfate, ammonium chloride, ammonium phosphate,ammonium carbonate and ammonium nitrate. The sources of nitrogen may beused individually or as a mixture. Suitable sources of phosphorusinclude phosphoric acid, potassium dihydrogen phosphate or dipotassiumhydrogen phosphate or the corresponding sodium-containing salts. Theculture medium must also contain salts of metals such as, e.g.,magnesium sulfate or iron sulfate which are necessary for growth.Finally, essential growth-promotors such as amino acids and vitamins maybe used in addition to the substances mentioned above. Moreover,suitable preliminary stages may be added to the culture medium. Thesubstances used may be added to the culture in the form of a singlepreparation or fed in a suitable manner during cultivation.

In order to control the pH of the culture, basic compounds such assodium hydroxide, potassium hydroxide, ammonia or ammoniacal gas liquoror acid compounds such as phosphoric acid or sulfuric acid may be usedin a suitable manner. Antifoaming agents such as, e.g., fatty acidpolyglycol esters may be used to control foam development. In order tomaintain the stability of plasmids, suitable selectively actingsubstances such as, e.g., antibiotics may be added to the medium. Tomaintain aerobic conditions, oxygen or oxygen-containing gas mixturessuch as, e.g., air may be introduced into the culture. The temperatureof the culture is normally from 20° C. to 45° C. and preferably from 25°C. to 40° C. The culture is continued until an L-lysine maximum hasformed. This objective is normally achieved within 10 hours to 160hours.

The invention also provides, therefore, a process for the fermentativepreparation of L-amino acids, particularly L-lysine, wherein thefollowing steps are carried out:

-   a) Fermentation of coryneform bacteria producing L-amino acids in    which at least the ptsH gene coding for component H of the    phosphotransferase system is enhanced, particularly overexpressed.-   b) Enrichment of the L-amino acid in the medium or in the cells of    the bacteria, and-   c) Isolation of the L-amino acid.

The analysis of L-lysine may be carried out by anion exchangechromatography followed by ninhydrin derivatisation, as described inSpackman et al. (Analytical Chemistry, 30, (1958), 1190).

The process according to the invention is used for the fermentativepreparation of L-amino acids, particularly L-lysine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in more detail below on the basis ofembodiments.

EXAMPLE 1

Preparation of a Genomic Cosmid Gene Bank from Corynebacteriumglutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolatedas described in Tauch et al. (1995, Plasmid 33:168–179) and partiallycleaved with the restriction enzyme Sau3AI (Amersham Pharmacia,Freiburg, Germany, product description Sau3AI, code no. 27-0913-02). TheDNA fragments were dephosphorylated with Shrimp alkaline phosphatase(Roche Molecular Biochemicals, Mannheim, Germany, product descriptionSAP, code no. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl etal. (1987) Proceedings of the National Academy of Sciences USA84:2160–2164), purchased from the company Stratagene (La Jolla, USA,product description SuperCos1 Cosmid Vector Kit, code no. 251301) wascleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg,Germany, product description XbaI, code no. 27-0948-02) and likewisedephosphorylated with Shrimp alkaline phosphatase. The cosmid DNA wasthen cleaved with the restriction enzyme BamHI (Amersham Pharmacia,Freiburg, Germany, product description BamHI, code no. 27-0868-04). Thecosmid DNA treated in this way was mixed with the treated ATCC 13032-DNAand the batch was treated with T4-DNA-ligase (Amersham Pharmacia,Freiburg, Germany, product description T4-DNA-Ligase, codeno.27-0870-04). The ligation mixture was then packaged into phages usingGigapack II XL Packing Extracts (Stratagene, La Jolla, USA, productdescription Gigapack II XL Packing Extract, code no. 200217). In orderto infect the E. coli strain NM554 (Raleigh et al. 1988, Nucleic AcidResearch 16:1563–1575) the cells were taken up in 10 mM MgSO₄ and mixedwith an aliquot of the phage suspension. Infection and titration of thecosmid bank were carried out as described in Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), the cellsbeing plated on LB-Agar (Lennox, 1955, Virology, 1:190) with 100 μg/mlampicillin. After incubation overnight at 37° C., recombinant individualclones were selected.

EXAMPLE 2

Isolation and Sequencing of the ptsH Gene

The cosmid DNA of an individual colony was isolated with the QiaprepSpin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) inaccordance with the manufacturer's instructions and partially cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, product description Sau3AI, product No. 27-0913-02). The DNAfragments were dephosphorylated with Shrimp alkaline phosphatase (RocheMolecular Biochemicals, Mannheim, Germany, product description SAP,product No. 1758250). After separation by gel electrophoresis, isolationof the cosmid fragments in the size region from 1500 to 2000 bp wascarried out with the QiaExII Gel Extraction Kit (product No. 20021,Qiagen, Hilden, Germany). The DNA of the sequencing vector pZero-1purchased from the company Invitrogen (Groningen, the Netherlands,product description Zero Background Cloning Kit, product No. K2500-01)was cleaved with the restriction enzyme BamHI (Amersham Pharmacia,Freiburg, Germany, product description BamHI, product No. 27-0868-04).Ligation of the cosmid fragments into the sequencing vector pZero-1 wascarried out as described by Sambrook et al. (1989, Molecular Cloning: Alaboratory Manual, Cold Spring Harbor), the DNA mixture being incubatedovernight with T4-ligase (Pharmacia Biotech, Freiburg, Germany). Thisligation mixture was then inserted in the E. coli strain DH5αMCR bymicroporation (Grant, 1990, Proceedings of the National Academy ofSciences U.S.A., 87:4645–4649)(Tauch et al. 1994, FEMS MicrobiolLetters, 123:343–7) and plated on LB-agar (Lennox, 1955, Virology,1:190) with 50 μg/ml Zeocin. Plasmid preparation of the recombinantclones was carried out with the Biorobot 9600 (Product No. 900200,Qiagen, Hilden, Germany). Sequencing was carried out by thedideoxy-chain termination method of Sanger et al. (1977, Proceedings ofthe National Academy of Sciences U.S.A., 74:5463–5467) withmodifications after Zimmermann et al. (1990, Nucleic Acids Research,18:1067). The “RR dRhodamin Terminator Cycle Sequencing Kit” from PEApplied Biosystems (product No. 403044, Weiterstadt, Germany) was used.Separation by gel electrophoresis and analysis of the sequencingreaction was carried out in a “Rotiphoresis NF acrylamide/bisacrylamide”gel (29:1) (product No. A124.1, Roth, Karlsruhe, Germany) with the “ABIPrism 377” sequencing device from PE Applied Biosystems (Weiterstadt,Germany).

The raw sequence data obtained were then processed using the Stadenprogram package (1986, Nucleic Acids Research, 14:217–231) version 97-0.The individual sequences of the pzero1 derivatives were assembled to acoherent contig. The computer-controlled coding region analysis wasprepared with the program XNIP (Staden, 1986, Nucleic Acids Research,14:217–231). Further analyses were carried out with the “BLAST searchprograms” (Altschul et al., 1997, Nucleic Acids Research, 25:3389–3402),against the non-redundant data base of the “National Center forBiotechnology Information” (NCBI, Bethesda, Md., USA).

The nucleotide sequence obtained is shown in SEQ ID no.1. The analysisof the nucleotide sequence revealed an open reading frame of 267 basepairs, which was designated the ptsH gene. The ptsH gene codes for aprotein of 89 amino acids.

EXAMPLE 3

Preparation of a Shuttle Vector pEC-K18mob2ptsHexp in Order to Enhancethe ptsH Gene in C. glutamicum

3.1 Cloning the ptsH Gene into the Vector pCR®Blunt II

Chromosomal DNA was isolated from the ATCC 13032 strain according to themethod of Eikmanns et al. (Microbiology 140: 1817–1828 (1994)). On thebasis of the sequence of the ptsH gene known from Example 2 for C.glutamicum, the following oligonucleotides were selected for thepolymerase chain reaction:

PtsHexp1 5′ - ACC ACT GGT GCA ATC TCC AT 3′ (SEQ ID NO:5) ptsHexp2 5′ -TTT ACT CAG CGT CAA GGT CC 3′ (SEQ ID NO:6)

The primers shown were synthesised by ARK Scientific GmbH Biosystems(Darmstadt, Germany) and the PCR reaction was carried out according tothe standard PCR method of Innis et al. (PCR protocols. A Guide toMethods and Applications, 1990, Academic Press) with Pwo-polymerase fromRoche Diagnostics GmbH (Mannheim, Germany). With the aid of thepolymerase chain reaction, the primers permit the amplification of a 686bp DNA-fragment which bears the ptsH gene with the potential promotorregion. The DNA sequence of the amplified DNA fragment was analysed bysequencing.

The amplified DNA fragment was ligated with the Zero Blunt™ Kit fromInvitrogen Corporation (Carlsbad, Calif., USA; catalogue numberK2700-20) into the vector pCR®Blunt II (Bernard et al., Journal ofMolecular Biology, 234: 534–541 (1993)).

The E. coli strain TOP10 was then electroporated with the ligation mix(Hanahan, In: DNA Cloning. A Practical Approach. Vol. I., IRL-Press,Oxford, Washington D.C., USA, 1985). The plasmid-bearing cells wereselected by plating the transformation mix onto LB agar (Sambrook etal., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) which had beensupplemented with 25 mg/l of kanamycin. Plasmid DNA was isolated from atransformant using the QIAprep Spin Miniprep Kit from Qiagen andanalysed by restriction with the restriction enzyme EcoRI followed byagarose gel electrophoresis (0.8%). The plasmid was named pCRB1-ptsHexpand is shown in FIG. 1.

3.2 Preparation of the E. coli—C. glutamicum Shuttle Vector pEC-K18mob2

The E. coli—C. glutamicum shuttle vector was constructed according tothe prior art. The vector contains the replication region rep of plasmidpGA1 including the replication effector per (U.S. Pat. No. 5,175,108;Nesvera et al., Journal of Bacteriology 179, 1525–1532 (1997)), thekanamycin resistance-conferring aph(3′)-IIa gene of the transposon Tn5(Beck et al., Gene 19, 327–336 (1982)), the replication region oriV ofthe plasmid pMB1 (Sutcliffe, Cold Spring Harbor Symposium onQuantitative Biology 43, 77–90 (1979)), the lacZα gene fragmentincluding the lac promotor and a multiple cloning site (mcs) (Norrander,J. M. et al., Gene 26, 101–106 (1983)) and the mob region of plasmid RP4(Simon et al., Biol/Technology 1: 784–791 (1983)). The vectorconstructed was transformed into the E. coli strain DH5αmcr (Hanahan,in: DNA Cloning. A Practical Approach. Vol. I, IRL-Press, Oxford,Washington D.C., USA). The plasmid-bearing cells were selected byplating the transformation mix onto LB agar (Sambrook et al., MolecularCloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) which had been supplemented with 25mg/l of kanamycin. Plasmid DNA was isolated from a transformant usingthe QIAprep Spin Miniprep Kit from Qiagen and analysed by restrictionwith the restriction enzyme EcoRI and HindIII followed by agarose gelelectrophoresis (0.8%). The plasmid was named pEC-K18mob2 and is shownin FIG. 2.

The following microorganism was deposited at the German Collection forMicroorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) inaccordance with the Budapest Agreement:

-   C.glutamicum strain DMS 5715/pEC-K18mob2 as DSM 13245

3.3 Cloning ptsH into the E. coli—C.glutamicum Shuttle VectorpEC-K18mob2

In order to clone the ptsH gene into the E. coli—C. glutamicum shuttlevector pEC-K18mob2 described in Example 3.2, plasmid DNA frompEC-K18mob2 was completely digested with the restriction endonucleasesKpnI and XbaI and treated with alkaline phosphatase (Alkalinephosphatase, Roche Diagnostics GmbH, Mannheim, Germany).

The vector pCRB1-ptsHexp was isolated from Escherichia coli Top10 andcompletely digested with the restriction endonucleases KpnI and XbaI,and the 788 bp fragment with the ptsH gene was purified from a 0.8%agarose gel (QIAquick Gel Extraction Kit from Qiagen, Hilden, Germany).The fragment with the ptsH gene was then ligated with the vectorpEC-K18mob2 (T4-ligase, Roche Diagnostics GmbH, Mannheim; Germany). Theligation mix was transformed into the E. coli strain DH5αmcr (Hanahan,in: DNA Cloning. A Practical Approach. Vol. I. IRL-Press, Oxford,Washington D.C., USA). The plasmid-bearing cells were selected byplating the transformation mix onto LB agar (Sambrook et al., MolecularCloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) which had been supplemented with25 mg/l of kanamycin. Plasmid DNA was isolated from a transformant usingthe QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany) and analysedby treatment with the restriction enzyme EcoRI followed by agarose gelelectrophoresis. The plasmid was named pEC-K18mob2ptsHexp and is shownin FIG. 3.

The strain was named E. coli DH5αmcr/pEC-K18mob2ptsHexp and deposited inthe form of a pure culture on 28 Nov. 2000 at the German Collection forMicroorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) as DSM13878, in accordance with the Budapest Agreement.

EXAMPLE 4

Transformation of the Strain DSM5715 with Plasmid pEC-K18mob2ptsHexp

The strain DSM5715 was transformed with plasmid pEC-K18mob2ptsHexp usingthe electroporation method described by Liebl et al., (FEMS MicrobiologyLetters, 53:299–303 (1989)). The transformants were selected on LBHISagar composed of 18.5 g/l brain-heart infusion broth, 0.5 M sorbitol, 5g/l Bacto-trypton, 2.5 g/l Bacto-yeast extract, 5 g/l NaCl and 18 g/lBacto-agar which had been supplemented with 25 mg/l kanamycin.Incubation took place for 2 days at 33° C.

Plasmid DNA was isolated from a transformant by the usual methods(Peters-Wendisch et al., 1998, Microbiology, 144, 915–927), cut with therestriction endonuclease EcoRI and the plasmid was then analysed byagarose gel electrophoresis. The strain obtained was namedDSM5715/pEC-K18mob2ptsHexp.

EXAMPLE 5

Preparation of Lysine

The C. glutamicum strain DSM5715/pEC-K18mob2ptsHexp obtained in Example4 was cultured in a nutrient medium suitable for the production oflysine, and the lysine content in the culture supernatant wasdetermined.

To this end, the strain was initially incubated for 24 hours at 33° C.on an agar plate with the appropriate antibiotic (brain-heart agar withkanamycin (25 mg/l)). Starting from this agar plate culture, apre-culture was inoculated (10 ml of medium in 100 ml Erlenmeyer flask).The medium used for the pre-culture was the solid medium Cg III.

Cg III medium NaCl 2.5 g/l Bacto-peptone  10 g/l Bacto-yeast extract  10g/l Glucose (autoclaved separately) 2% (w/v) The pH was adjusted to 7.4

Kanamycin (25 mg/l) was added thereto. The pre-culture was incubated for16 hours at 33° C. at 240 rpm on the shaker. A main culture wasinoculated from this pre-culture, so that the initial OD (660 nm)of themain culture was 0.05. MM medium was used for the main culture.

MM medium CSL (Corn Steep Liquor) 5 /gl MOPS (morpholinopropane sulfonicacid) 20 g/l Glucose (autoclaved separately) 100 g/l (NH₄)₂SO₄ 25 g/lKH₂PO₄ 0.1 g/l MgSO₄ * 7 H₂O 1.0 g/l CaCl₂ * 2 H₂O 10 mg/l FeSO₄ * 7 H₂O10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin (filter-sterilised) 0.3 mg/lThiamine * HCl (filter-sterilised) 0.2 mg/l L-leucine(filter-sterilised) 0.1 g/l CaCO₃ 25 g/l

CSL, MOPS and the salt solution were adjusted to pH 7 with ammoniasolution and autoclaved. The sterile substrate and vitamin solutionswere then added, and the dry-autoclaved CaCO₃.

The culture was carried out in 10 ml volumes in a 100 ml Erlenmeyerflask with baffles. Kanamyin (25 mg/l) was added. The culture wascarried out at 33° C. and at 80% air humidity.

After 48 hours and 72 hours the OD was determined at a measuringwavelength of 660 nm with the Biomek 1000(Beckmann Instruments GmbH,Munich). The amount of lysine formed was determined with an amino acidanalyser from Eppendorf-Biotronik (Hamburg, Germany) by ion exchangechromatography and post-column derivatisation with ninhydrin detection.

The result of the test is shown in Table 1.

TABLE 1 Lysine-HCl Strain OD (660 nm) g/l DSM5715/pEC-K18mob2 11.4 14.14(48 hours) DSM5715/pEC-K18mob2ptsHexp 10.7 15.98 (48 hours)DSM5715/pEC-K18mob2mob2 10.1 15.24 (72 hours) DSM5715/pEC-K18mob2ptsHexp10.0 17.13 (72 hours)

The following Figures are attached:

FIG. 1: Map of plasmid pCRB1-ptsHexp

FIG. 2: Map of plasmid pEC-K18mob2

FIG. 3: Map of plasmid pEC-K18mob2ptsHexp

The abbreviations and names used have the following meaning: Kan:resistance gene for kanamycin Zeocin: Zeocin resistance gene ptsH: ptsHgene from C. glutamicum ColE1: Replication origin of plasmid CelE1lacZ-alpha: lacZ gene fragment from E. coli lacZ-alpha{grave over ( )}:fragment of the lacZ gene fragment from E. coli per: gene forcontrolling the copy number from pGA1 oriV: ColE1-like origin from pMB1rep: plasmid-coded replication region from C. glutamicum plasmid pGA1RP4mob: RP4 mobilisation site EcoRI: restriction site of the restrictionenzyme EcoRI HindIII: restriction site of the restriction enzyme HindIIIKpnI: restriction site of the restriction enzyme KpnI XbaI: restrictionsite of the restriction enzyme XbaI

1. A process for the production of L-lysine comprising: culturing a coryneform bacterium in which there is overexpression of a polynucleotide selected from the group consisting of: i) a polynucleotide having the sequence as set forth in SEQ ID NO: 1; ii) a polynucleotide encoding a phosphocarrier component H polypeptide of a phosphotransferase system comprising an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 2; iii) a polynucleotide encoding the amino acid sequence as set forth in SEQ ID NO: 2; iv) a polynucleotide having the sequence as set forth in SEQ ID NO: 3; and v) a polynucleotide encoding the amino acid sequence as set forth in SEQ ID NO: 4; in a medium suitable for expression of said polynucleotide, wherein overexpression occurs by increasing the copy number of said polynucleotide or operatively linking said polynucleotide to a promoter.
 2. The process according to claim 1, further comprising: a) concentrating the L-lysine produced, in the medium or in the cells of the bacterium; and b) isolating the L-lysine produced.
 3. The process according to claim 1, wherein one or more C. glutamicum genes selected from the group consisting of: (a) the dapA gene which codes for dihydrodipicopicolinate synthase, (b) the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase, (c) the tpi gene which codes for triose phosphate isomerase, (d) the pgk gene which codes for 3-phosphoglycerate kinase, (e) the ptsM gene which codes for component M of the phosphoenolpyruvate-sugar-phosphotransferase system, (f) the pyc gene which codes for pyruvate carboxylase, and (g) the lysE gene which codes a L-lysine export carrier, is overexpressed by increasing the copy number or placing said gene under a strong promoter during fermentation for the preparation of L-lysine.
 4. The process according to claim 1, wherein expression of one or more C. glutamicum genes selected from the group consisting of: (a) the pck gene which codes for phosphoenol pyruvate carboxykinase, (b) the pgi gene which codes for glucose 6-phosphate isomerase, and (c) the poxB gene which codes for pyruvate oxidase, is or arc attenuated.
 5. The process according to claim 1, wherein the coryneform bacterium is Corynebacterium glutamicum.
 6. The process according to claim 1, wherein the coryneform bacterium is transformed with a vector comprising the overexpressed polynucleotide.
 7. The process according to claim 1, wherein the coryneform bacterium is C. glutamicum strain DSM 5715 that is transformed with the vector pEC-K18mob2ptsHexp as shown in FIG.
 3. 8. A process for producing L-lysine comprising the following steps: (a) transforming a coryneform bacterium with plasmid vector pEC-K18mob2ptHexp as shown in FIG. 3; (b) culturing said bacterium in a medium suitable for producing said L-lysine; and (c) isolating L-lysine.
 9. The process according to claim 8, wherein the coryneform bacterium is Corynebacterium glutamicum.
 10. The process according to claim 9, wherein the coryneform bacterium is a bacterium of C. glutamicum strain DSM
 5715. 11. The process according to claim 9, wherein the expression of a ptsH polynucleotide comprising the nucleotide sequence as set forth in SEQ ID NO: 1 is overexpressed during fermentation for the preparation of L-lysine by increasing the copy number or operatively linking said ptsH polynucleotide to a strong promoter. 